The present invention relates to the finding of a novel interaction between the VHL and HIF proteins, assays based upon this interaction and novel compounds obtainable by such assay methods.
Enhanced glucose metabolism and angiogenesis are classical features of cancer involving up-regulation of genes which are normally inducible by hypoxia. In addition to stimulation by the hypoxic microenvironment, genetic alterations contribute to these effects. A striking example is von Hippel-Lindau (VHL) disease, a hereditary human cancer syndrome predisposing to highly angiogenic tumours, particularly of the central nervous system, kidney, retina and adrenal glands.
VHL syndrome is caused by germline mutations in the VHL tumour suppressor, and VHL tumours are associated with loss or mutation of the remaining wild-type allele. VHL is also inactivated in xe2x80x9480% of sporadic clear cell renal carcinomas (RCC), the predominant form of kidney cancer. The ability of RCC cells to form tumours in nude mice can be abrogated by introduction of wild-type VHL.
VHL-associated tumours are highly vascularized, and this supports the current model that VHL negatively regulates the production of hypoxia-inducible factors such as the angiogenic vascular endothelial growth factor (VEGF). VHLxe2x88x92/xe2x88x92 tumour cells have high levels of these factors, and reintroduction of VHL down-regulates them under normoxic conditions. The mechanism of this VHL activity is not well understood.
Stebbins et al (Science, 1999, 284; 55-61) report that the VHL protein forms a complex with the Elongin C and Elongin B proteins, and that the complex (the VCB complex) is formed by a direct interaction of VHL and Elongin C, with a second interaction between the Elongin C and B proteins. The interface of VHL which interacts with Elongin C contains a number of residues which are commonly mutated in VHL syndrome. The authors also found a second domain of VHL, not involved in binding Elongin C, which they speculate may correspond to another macromolecular binding site of VHL.
Hypoxia inducible factor-1 (HIF-1) plays a key role in a wide variety of cellular responses to hypoxia, including the regulation of genes involved in energy metabolism, vasomotor control, angiogenesis, proliferation, apoptosis and matrix remodelling. HIF is a heterodimer of an HIF xcex1 subunit and the aryl hydrocarbon receptor nuclear translocator (ARNT) protein, a member of the PAS superfamily of basic helix-loop-helix proteins. A major regulatory mechanism involves proteolysis of HIF xcex1 subunits which are rapidly degraded by the proteasome in normoxia but stabilised by hypoxia.
Although it has been proposed in the past that the VHL protein may mediate its effect via the destabilisation of hypoxia-regulated mRNA transcripts, we have surprisingly found that this protein has a direct interaction with HIF xcex1 subunits. While not wishing to be bound by any one theory, it is believed that the formation of a VHL-xcex1 subunit complex targets the xcex1 subunits for destruction, possibly by the association of the complex with the Elongin B subunit, which has homology to ubiquitin.
Modulation of the interaction of VHL with HIF xcex1 subunits has a variety of uses. Blocking the interaction may facilitate cell cycle progression and the production of a number of proteins which promote angiogenesis and/or promote cellular survival or cellular function in hypoxia, a desirable outcome in the treatment of certain clinical conditions, particularly ischaemic conditions such as coronary, cerebral and vascular insufficiency.
Thus in a first aspect the present invention provides an assay for a modulator of VHL-HIF xcex1 subunit interaction, which comprises:
a) bringing into contact a VHL protein, a HIF xcex1 subunit protein and a putative modulator compound under conditions where the VHL protein and the HIF xcex1 subunit protein, in the absence of modulator, are capable of forming a complex; and
b) measuring the degree of inhibition of complex formation caused by said modulator compound.
The present invention further provides an assay for a modulator of VHL-HIF xcex1 subunit interaction, which comprises:
a) bringing into contact a VHL protein, a HIF xcex1 subunit protein and a putative modulator compound under conditions where the VHL protein and the HIF xcex1 subunit protein, in the absence of modulator, are capable of forming a complex;
b) providing an HIF response element to which the HIF xcex1 subunit protein is capable of binding and/or transcriptionally activating; and
c) measuring the degree of modulation of binding of the xcex1 subunit to, or transcriptional activation of, the response element caused by said modulator compound.
In a further aspect, the invention provides compounds obtainable by such an assays for example peptide compounds based on the portions of VHL or HIF xcex1 subunit which interact with each other.
The assay of the invention may be performed in vitro using isolated, purified or partially purified VHL and HIF xcex1 subunit proteins, or in cell free or cellular systems in which case the assay may optionally be performed in the presence of a factor which promotes a normoxic cellular state, such as the presence of oxygen (e.g. about 21% O2, 5% CO2, balance N2) and/or the presence of hydrogen peroxide at about 50-200 xcexcM.